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Peptide Mixtures (peptide + mixture)
Kinds of Peptide Mixtures Selected AbstractsA Novel MALDI Matrix for Analyzing Peptides and Proteins: Paraffin Wax Immobilized Matrix,CHINESE JOURNAL OF CHEMISTRY, Issue 1 2009Yuanlong WEI Abstract A new kind of MALDI matrix, termed paraffin wax immobilized matrix, was used to study peptide mixtures and proteins. During the preparation process, the paraffin wax was heated and coated on the stainless-steel target plate, and then 2,5-dihydrobenzoic acid (DHB) was deposited on the paraffin layer and stainless-steel target plate to obtain different kinds of matrix spots. The morphology of matrices on different supports and peptide-matrix co-crystallization were observed by a high resolution digital-video microscopy system. Peptide mixtures and bovine serum albumin (BSA) digests were used to investigate the performance of the immobilized matrices on the paraffin target. The MALDI-FTMS analysis results also showed that the detection sensitivity of matrices immobilized in the paraffin sample support was better than that on other sample supports. [source] High-sensitivity analysis of specific peptides in complex samples by selected MS/MS ion monitoring and linear ion trap mass spectrometry: Application to biological studiesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 11 2007Inmaculada Jorge Abstract Mass spectrometry (MS) is a technique of paramount importance in Proteomics, and developments in this field have been possible owing to novel MS instrumentation, experimental strategies, and bioinformatics tools. Today it is possible to identify and determine relative expression levels of thousands of proteins in a biological system by MS analysis of peptides produced by proteolytic digestion. In some situations, however, the precise characterization of a particular peptide species in a very complex peptide mixture is needed. While single-fragment ion-based scanning modes such as selected ion reaction monitoring (SIRM) or consecutive reaction monitoring (CRM) may be highly sensitive, they do not produce MS/MS information and their actual specificity must be determined in advance, a prerequisite that is not usually met in a basic research context. In such cases, the MS detector may be programmed to perform continuous MS/MS spectra on the peptide ion of interest in order to obtain structural information. This selected MS/MS ion monitoring (SMIM) mode has a number of advantages that are fully exploited by MS detectors that, like the linear ion trap, are characterized by high scanning speeds. In this work, we show some applications of this technique in the context of biological studies. These results were obtained by selecting an appropriate combination of scans according to the purpose of each one of these research scenarios. They include highly specific identification of proteins present in low amounts, characterization and relative quantification of post-translational modifications such as phosphorylation and S -nitrosylation and species-specific peptide identification. Copyright © 2007 John Wiley & Sons, Ltd. [source] New approach for rapid detection of known hemoglobin variants using LC-MS/MS combined with a peptide database,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2007F. Basilico Abstract The identification of hemoglobin (Hb) variants is usually performed by means of different analytical steps and methodologies. Phenotypic methods, such as gel electrophoresis and high performance liquid chromatography, are used to detect the different electrophoretic or chromatographic behaviors of hemoglobin variants in comparison to HbA0 used as a control. These data often need to be combined with mass spectrometry analyses of intact globins and their tryptic peptide mixtures. As an alternative to a ,step-by-step' procedure, we have developed a ,single step' approach for the identification of Hb variants present in biological samples. This is based on the µHPLC-ESI-MS/MS analysis of the peptide mixture generated by a tryptic digestion of diluted Hb samples and an in-house new database containing solely the variant tryptic peptide of known human Hb variants. The experimental results (full MS and MS/MS spectra) are correlated with theoretical mass spectra generated from our in-house-built variant peptide database (Hbp) using the SEQUEST algorithm. Simple preparation of samples and an automated identification of the variant peptide are the main characteristics of this approach, making it an attractive method for the detection of Hb variants at the routine clinical level. We have analyzed 16 different samples, each containing a different known variant of hemoglobin. Copyright © 2006 John Wiley & Sons, Ltd. [source] Preparative separation of a multicomponent peptide mixture by mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 2 2006Xinli Yang Abstract We report on the first multiplex preparative separation by mass spectrometry of bio-organic molecules in the 200,350 Da mass range that is typical for synthetic drugs. A five-component mixture consisting of two di- and three tripeptides has been separated by mass using a specially designed mass spectrometer. The instrument for preparative separations consists of an electrospray ionization (ESI) source, ion transfer optics, an electrostatic sector, and an inhomogeneous-field magnetic mass analyzer that achieves linear mass dispersion of ion beams. Protonated peptides produced by electrospray were separated, nondestructively landed on a 16-channel array of dry collector plates, and reconstituted in solution. The preparation procedures and the instrumental conditions have been optimized to maximize the ion currents. The significant features of the special mass spectrometer are high ion currents and simultaneous separation and collection of mixture components. Copyright © 2006 John Wiley & Sons, Ltd. [source] Effective detection of peptides containing cysteine sulfonic acid using matrix-assisted laser desorption/ionization and laser desorption/ionization on porous silicon mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2006Tomoya Kinumi Abstract Cysteine sulfonic acid-containing peptides, being typical acidic peptides, exhibit low response in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, matrix conditions and the effect of diammonium hydrogencitrate (DAHC) as additive were investigated for ionization of cysteine sulfonic acid-containing peptides in MALDI. A matrix-free ionization method, desorption/ionization on porous silicon (DIOS), was also utilized to evaluate the effect of DAHC. When equimolar three-component mixtures of peptides carrying free cysteine, cysteine sulfonic acid, and carbamidomethyl cysteine were measured by MALDI using a common matrix, ,-cyano-4-hydroxycinnamic acid (CHCA), no signal corresponding to cysteine sulfonic acid-containing peptide could be observed in the mass spectrum. However, by addition of DAHC to CHCA, the peaks of cysteine sulfonic acid-containing peptides were successfully observed, as well as when using 2,4,6-trihydroxyacetophenone (THAP) and 2,6-dihydroxyacetophenone with DAHC. In the DIOS mass spectra of these analytes, the use of DAHC also enhanced the peak intensity of the cysteine sulfonic acid-containing peptides. On the basis of studies with these model peptides, tryptic digests of oxidized peroxiredoxin 6 were examined as a complex peptide mixture by MALDI and DIOS. In MALDI, the peaks of cysteine sulfonic acid-containing peptides were observed when using THAP/DAHC as the matrix, but this was not so with CHCA. In DIOS, the signal from cysteine sulfonic acid-containing peptides was suppressed; however, the use of DAHC significantly enhanced the signal intensity with an increase in the number of observed peptides and increased signal-to-noise ratio in the DIOS spectra. The results show that DAHC in the matrix or on the DIOS chip decreases discrimination and suppression effects in addition to suppressing alkali-adduct ions, which leads to a beneficial effect on protonation of peptides containing cysteine sulfonic acid. Copyright © 2005 John Wiley & Sons, Ltd. [source] Sequence diversity of the peptaibol antibiotic suzukacillin-A from the mold Trichoderma virideJOURNAL OF PEPTIDE SCIENCE, Issue 5 2006Corina Krause Abstract From the culture broth of the mold Trichoderma viride, strain 63 C-I, the polypeptide antibiotic suzukacillin (SZ) was isolated. A peptide mixture named SZ-A was obtained by crystallization from crude SZ. Individual peptides from SZ-A were isolated by semipreparative HPLC and sequences were determined by HPLC-ESI-MS. The data confirm a general sequence of SZ-A published previously and in addition establish the individual sequences of 15 acetylated eicosa peptides with C -terminal alcohols. The major peptide SZ-A4 (21% of all peptides) shows the sequence: Ac-Aib-Ala-Aib-Ala-Aib-Ala6 -Gln-Aib-Lx9 -Aib-Gly-Aib12 -Aib-Pro-Vx15 -Aib-Vx17 -Gln-Gln-Fol. Amino acid exchanges of the peptaibol are located in position 6 (Ala/Aib), 9 (Vx/Lx), 12 (Aib/Lx), 17 (Aib/Vx) and possibly at position15 (Val/Iva) (uncommon abbreviations: Aib (,-aminoisobutyric acid); Iva (D -isovaline); Lx (L -leucine or L -isoleucine); Vx (L -valine or D -isovaline); Fol (L -phenylalaninol)). Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd. [source] Bioaffinity magnetic reactor for peptide digestion followed by analysis using bottom-up shotgun proteomics strategyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 3 2008Lucie Korecká Abstract We report an efficient and streamlined way to improve the analysis and identification of peptides and proteins in complex mixtures of soluble proteins, cell lysates, etc. By using the shotgun proteomics methodology combined with bioaffinity purification we can remove or minimize the interference contamination of a complex tryptic digest and so avoid the time-consuming separation steps before the final MS analysis. We have proved that by means of enzymatic fragmentation (endoproteinases with Arg-C or/and Lys-C specificity) connected with the isolation of specific peptides we can obtain a simplified peptide mixture for easier identification of the entire protein. A new bioaffinity sorbent was developed for this purpose. Anhydrotrypsin (AHT), an inactive form of trypsin with an affinity for peptides with arginine (Arg) or lysine (Lys) at the C-terminus, was immobilized onto micro/nanoparticles with superparamagnetic properties (silica magnetite particles (SiMAG),Carboxyl, Chemicell, Germany). This AHT carrier with a determined binding capacity (26.8 nmol/mg of carrier) was tested with a model peptide, human neurotensin, and the resulting MS spectra confirmed the validity of this approach. [source] Gel-free sample preparation for the nanoscale LC-MS/MS analysis and identification of low-nanogram protein samplesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 14 2007Marco Gaspari Abstract Protein identification at the low nanogram level could in principle be obtained by most nanoscale LC-MS/MS systems. Nevertheless, the complex sample preparation procedures generally required in biological applications, and the consequent high risk of sample losses, very often hamper practical achievement of such low levels. In fact, the minimal amount of protein required for the identification from a gel band or spot, in general, largely exceeds the theoretical limit of identification reachable by nanoscale LC-MS/MS systems. A method for the identification of low levels of purified proteins, allowing limits of identification down to 1 ng when using standard bore, 75 ,m id nanoscale LC-MS/MS systems is here reported. The method comprises an offline two-step sample cleanup, subsequent to protein digestion, which is designed to minimize sample losses, allows high flexibility in the choice of digestion conditions and delivers a highly purified peptide mixture even from "real world" digestion conditions, thus allowing the subsequent nanoscale LC-MS/MS analysis to be performed in automated, unattended operation for long series. The method can be applied to the characterization of low levels of affinity purified proteins. [source] MapQuant: Open-source software for large-scale protein quantificationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2006Kyriacos C. Leptos Abstract Whole-cell protein quantification using MS has proven to be a challenging task. Detection efficiency varies significantly from peptide to peptide, molecular identities are not evident a,priori, and peptides are dispersed unevenly throughout the multidimensional data space. To overcome these challenges we developed an open-source software package, MapQuant, to quantify comprehensively organic species detected in large MS datasets. MapQuant treats an LC/MS experiment as an image and utilizes standard image processing techniques to perform noise filtering, watershed segmentation, peak finding, peak fitting, peak clustering, charge-state determination and carbon-content estimation. MapQuant reports abundance values that respond linearly with the amount of sample analyzed on both low- and high-resolution instruments (over a 1000-fold dynamic range). Background noise added to a sample, either as a medium-complexity peptide mixture or as a high-complexity trypsinized proteome, exerts negligible effects on the abundance values reported by MapQuant and with coefficients of variance comparable to other methods. Finally, MapQuant's ability to define accurate mass and retention time features of isotopic clusters on a high-resolution mass spectrometer can increase protein sequence coverage by assigning sequence identities to observed isotopic clusters without corresponding MS/MS data. [source] Global phosphoproteome analysis on human HepG2 hepatocytes using reversed-phase diagonal LCPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 14 2005Kris Gevaert Professor Abstract We present a phosphoproteomics approach using diagonal RP chromatography as the basic isolation principle. Phosphopeptides present in a tryptic digest of total cellular lysates were first enriched by Fe3+ -immobilized metal ion affinity chromatography. Further sorting of the phosphopeptides took place in three steps. First, the resulting peptide mixture was fractionated over reversed-phase chromatography. Second, peptides present in each fraction were treated with phosphatases. Third, the dephosphorylated peptides were then more hydrophobic and shifted towards a later elution interval from the contaminating non-phosphopeptides eluting at the same position as during the primary run. Since the phosphopeptides are isolated as their dephosphorylated form, additional proof for their original phosphorylation state was obtained by split-differential 16O ,18O labeling. The method was validated with alpha-casein phosphopeptides and consecutively applied on HepG2 cells. We identified 190,phosphorylated peptides from 152,different proteins. This dataset includes 38,novel protein phosphorylation sites. [source] The human plasma proteome: Analysis of Chinese serum using shotgun strategyPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 13 2005Ping He Abstract We have investigated the serum proteome of Han-nationality Chinese by using shotgun strategy. A complete proteomics analysis was performed on two reference specimens from a total of 20,healthy donors, in which each sample was made from ten-pooled male or female serum, respectively. The methodology used encompassed (1),removal of six high-abundant proteins; (2),tryptic digestion of low- and high-abundant proteins of serum; (3),separation of peptide mixture by RP-HPLC followed by ESI-MS/MS identification. A total of 944,nonredundant proteins were identified under a stringent filter condition (Xcorr,,,1.9, ,2.2, and ,3.75, ,Cn,,,0.1, and Rsp,,,4.0) in both pooled male and female samples, in which 594 and 622,entire proteins were found, respectively. Compared with the total 3020 protein identifications confirmed by more than one laboratory or more than one specimen in HUPO Plasma Proteome Project (PPP) participating laboratories recently, 206,proteins were identified with at least two distinct peptides per protein and 185,proteins were considered as high-confidence identification. Moreover, some lower abundance serum proteins (ng/mL range) were detected, such as complement,C5 and CA125, routinely used as an ovarian cancer marker in plasma and serum. The resulting nonredundant list of serum proteins would add significant information to the knowledge base of human plasma proteome and facilitate disease markers discovery. [source] The use of acetone as a substitute for acetonitrile in analysis of peptides by liquid chromatography/electrospray ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 1 2010Theodore R. Keppel The recent worldwide shortage of acetonitrile has prompted interest in alternative solvents for liquid chromatography/mass spectrometry (LC/MS). In this work, acetone was substituted for acetonitrile in the separation of a peptide mixture by reversed-phase high-performance liquid chromatography (RP-HPLC) and in the positive electrospray ionization mass spectrometry (ESI-MS) of individual peptides. On both C12 and C18 stationary phases, the substitution of acetone for acetonitrile as the organic component of the mobile phase did not alter the gradient elution order of a five-peptide retention standard, but did increase peak width, shorten retention times, and increase peak tailing. Positive ESI mass spectra were obtained for angiotensin I, bradykinin, [Leu5]-enkephalin, and somatostatin 14 dissolved in both acetonitrile/water/formic acid (25%/75%/0.1%) and acetone/water/formic acid (25%/75%/0.1%). Under optimized ESI-MS conditions, the mass spectral response of [Leu5]-enkephalin was increased two-fold when the solvent contained acetone. The substitution of acetone for acetonitrile resulted in only slight changes in the responses of the remaining peptides. A higher capillary voltage was required for optimum response when acetone was used. Compared with acetonitrile/water/formic acid (50/50/0.1%), more interfering species below m/z,=,140 were found in the ESI-MS spectra of acetone/water/formic acid (50/50/0.1%). Copyright © 2009 John Wiley & Sons, Ltd. [source] Coupling of ion-molecule reactions with liquid chromatography on a quadrupole ion trap mass spectrometerRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 8 2008Yuriy Pyatkivskyy We report for the first time a coupling of gas-phase ion-molecule reactions with chromatographic separations on a quadrupole ion trap mass spectrometer. The interface was accomplished by using a pulsed valve for the introduction of a volatile neutral into the ion trap. The pulsed valve controller is synchronized with the mass spectrometer software. The setup requires some minor modifications to the vacuum system of the commercial quadrupole ion trap but most of the modifications are external to the mass spectrometer. Two applications of this interface are described: differentiation between two phosphoglucose positional isomers and detection of a phosphopeptide in a peptide mixture. Both applications are using the reactivity of trimethoxyborate towards a phosphate moiety in the negative ion mode. The detection of phosphopeptides hinges on our findings that non-phosphorylated peptide anions do not react with trimethoxyborate. This LC/MS detection can be easily visualized in terms of selected reaction monitoring. Copyright © 2008 John Wiley & Sons, Ltd. [source] Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of 4-sulfophenyl isothiocyanate-derivatized peptides on AnchorChipÔ sample supports using the sodium-tolerant matrix 2,4,6-trihydroxyacetophenone and diammonium citrateRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2005Leon P. Oehlers The reagent 4-sulfophenyl isothiocyanate (SPITC) is an effective, stable, and inexpensive alternative to commercially available reagents used in the N-terminal sulfonation of peptides for enhanced postsource decay (PSD) in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric (MALDI-TOFMS) analyses. However, suppression of ionization of sulfonated peptides due to sample and matrix contaminants such as sodium can be a problem when using prestructured MALDI target sample supports, such as the Bruker Daltonics AnchorChipÔ. We show that use of the salt-tolerant matrix 2,4,6-trihydroxyacetophenone containing diammonium citrate (THAP/DAC) as an alternative to , -cyanohydroxycinnamic acid (HCCA) reduces the need for extensive washing of ZipTip-bound peptides or additional on-target sample clean-up steps. Use of the THAP/DAC matrix results in selective ionization of sulfonated peptides with greater peptide coverage, as well as detection of higher mass derivatized peptides, than was observed for HCCA or THAP alone. The THAP/DAC matrix is quite tolerant of sodium contamination, with SPITC-peptides detectable in preparations containing up to 50,mM NaCl. In addition, THAP/DAC matrix was found to promote efficient PSD fragmentation of sulfonated peptides. We demonstrated the utility of using the THAP/DAC MALDI matrix for peptide sequencing with DNA polymerase , tryptic peptide mixture, as well as tryptic peptides derived from Xiphophorus maculatus brain extract proteins previously separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Copyright © 2005 John Wiley & Sons, Ltd. [source] High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometryELECTROPHORESIS, Issue 21 2003Alexander R. Ivanov Abstract High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 ,m inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n -propanol and formamide as porogens and azobisisobutyronitrile as initiator. N -Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300,000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method. [source] New approach for rapid detection of known hemoglobin variants using LC-MS/MS combined with a peptide database,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 3 2007F. Basilico Abstract The identification of hemoglobin (Hb) variants is usually performed by means of different analytical steps and methodologies. Phenotypic methods, such as gel electrophoresis and high performance liquid chromatography, are used to detect the different electrophoretic or chromatographic behaviors of hemoglobin variants in comparison to HbA0 used as a control. These data often need to be combined with mass spectrometry analyses of intact globins and their tryptic peptide mixtures. As an alternative to a ,step-by-step' procedure, we have developed a ,single step' approach for the identification of Hb variants present in biological samples. This is based on the µHPLC-ESI-MS/MS analysis of the peptide mixture generated by a tryptic digestion of diluted Hb samples and an in-house new database containing solely the variant tryptic peptide of known human Hb variants. The experimental results (full MS and MS/MS spectra) are correlated with theoretical mass spectra generated from our in-house-built variant peptide database (Hbp) using the SEQUEST algorithm. Simple preparation of samples and an automated identification of the variant peptide are the main characteristics of this approach, making it an attractive method for the detection of Hb variants at the routine clinical level. We have analyzed 16 different samples, each containing a different known variant of hemoglobin. Copyright © 2006 John Wiley & Sons, Ltd. [source] Evaluation of the titanium dioxide approach for MS analysis of phosphopeptidesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 12 2006Clementine Klemm Abstract The affinity of titanium dioxide for phosphate groups has been successfully used for enrichment of phosphopeptides from complex mixtures. This paper reports the relationship between the occurrence of some amino acids and the phospho-specific and nonspecific binding of peptides that occurs during titanium dioxide enrichment. In order to perform a systematic study, two well-characterized peptide mixtures consisting of either 33 or 8 synthetic phosphopeptides and their nonphosphorylated analogs, which differed in charge and hydrophobicity, were synthesized and analyzed by ESI-MS and MALDI-MS. The titanium dioxide procedure was also evaluated for comprehensive detection of phosphopeptides in phosphoproteomics. In summary, our results clearly confirm the high selectivity of titanium dioxide for phosphorylated sequences. Drastically reduced recovery was observed for phosphopeptides with multiple basic amino acids. Nonspecific binding of nonphosphorylated peptides and sample loss of phosphopeptides must also be taken into account. Copyright © 2006 John Wiley & Sons, Ltd. [source] A novel class of chemically modified iodo-containing resins: design, synthesis and application to mass spectrometry-based proteome analysisJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 4 2004Li Zhang Abstract A novel class of chemically modified iodo-containing resins with isotope-labeled tagging for mass spectrometry-based proteome analysis is described. This iodo-containing resin contains a thiol-reactive group that is used to capture the cysteine (Cys)-containing peptides from peptide mixtures, one ,tag' amino acid, and an aminomethyl polystyrene resin with Rink Amide Linker. The ,tag' amino acid is synthesized in both heavy and light isotope-coded forms and therefore permits the direct relative quantification of peptides/proteins through mass spectrometric analysis. In the iodo-containing resin strategy, the Cys-containing peptides of two samples covalently captured by either light or heavy iodo-containing resin were mixed and washed extensively under stringent conditions. Then the Cys-containing peptides were retrieved by acid-catalyzed elution. Finally, the eluted peptides were directly analyzed by micro liquid chromatography/mass spectrometry for identification and relative quantification. The iodo-containing resins were synthesized by a simple but effective method. Their abilities to identify and quantify the Cys-containing part in two samples were proved by the analysis of mixtures of amino acids, peptides and proteins. Copyright © 2004 John Wiley & Sons, Ltd. [source] Assessing a novel microfluidic interface for shotgun proteome analysesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 10 2007An Staes Abstract Microfluidic interfaces coupled to ESI mass spectrometers hold great potential for proteomics as they have been shown to augment the overall sensitivity of measurements and require only a minimum of operator manipulations as compared to conventional nano-LC interfaces. Here, we evaluated a new type of HPLC-Chips holding larger enrichment columns (thus an increased sample loading capacity) for gel-free proteome studies. A tryptic digest of a human T-cell proteome was fractionated by strong cation exchange chromatography and selected fractions were analyzed by MS/MS on an IT mass spectrometer using both the new HPLC-Chip as well as a conventional nano-LC-MS/MS interface. Our results indicate that the HPLC-Chip is capable of handling very complex peptide mixtures and, in fact, leads to the identification of more peptides and proteins as compared to when a conventional interface was used. The HPLC-Chip preferentially produced doubly charged tryptic peptides. We further show that MS/MS spectra of doubly charged tryptic peptide ions are more readily identified by MASCOT as compared to those from triply charged precursors and thus argue that besides the improved chromatographic conditions provided by the HPLC-Chip, its peptide charging profile might be a secondary factor leading to an increased proteome coverage. [source] On-plate-selective enrichment of glycopeptides using boronic acid-modified gold nanoparticles for direct MALDI-QIT-TOF MS analysisPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 22 2009Jia Tang Abstract In this study, an on-plate-selective enrichment method is developed for fast and efficient glycopeptide investigation. Gold nanoparticles were first spotted and sintered on a stainless-steel plate, then modified with 4-mercaptophenylboronic acid to provide porous substrate with large specific surface and dual functions. These spots were used to selectively capture glycopeptides from peptide mixtures and the captured target peptides could be analyzed by MALDI-MS simply by deposition of 2,5-dihydroxybenzoic acid matrix. Horseradish peroxidase was employed as a standard glycoprotein to investigate the enrichment efficiency. In this way, the enrichment, washing and detection steps can all be fulfilled on a single MALDI target plate. The relatively small sample amount needed, low detection limit and rapid selective enrichment have made this on-plate strategy promising for online enrichment of glycopeptides, which could be applied in high-throughput proteome research. [source] Quantitative analysis of phosphopeptides in search of the disease biomarker from the hepatocellular carcinoma specimenPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 12 2009Hyoung-Joo Lee Abstract Reversible phosphorylation of proteins is the most common PTM in cell-signaling pathways. Despite this, high-throughput methods for the systematic detection, identification, and quantification of phosphorylated peptides have yet to be developed. In this paper, we describe the establishment of an efficient online titaniuim dioxide (TiO2)-based 3-D LC (strong cationic exchange/TiO2/C18)-MS3 -linear ion trap system, which provides fully automatic and highly efficient identification of phosphorylation sites in complex peptide mixtures. Using this system, low-abundance phosphopeptides were isolated from cell lines, plasma, and tissue of healthy and hepatocellular carcinoma (HCC) patients. Furthermore, the phosphorylation sites were identified and the differences in phosphorylation levels between healthy and HCC patient specimens were quantified by labeling the phosphopeptides with isotopic analogs of amino acids (stable isotope labeling with amino acids in cell culture for HepG2 cells) or water (HO for tissues and plasma). Two examples of potential HCC phospho-biomarkers including plectin-1(phopho-Ser-4253) and alpha-HS-glycoprotein (phospho-Ser 138 and 312) were identified by this analysis. Our results suggest that this comprehensive TiO2 -based online-3-D LC-MS3 -linear ion trap system with high-throughput potential will be useful for the global profiling and quantification of the phosphoproteome and the identification of disease biomarkers. [source] Cysteine-reactive covalent capture tags for enrichment of cysteine-containing peptidesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2009Priscille Giron Considering the tremendous complexity and the wide dynamic range of protein samples from biological origin and their proteolytic peptide mixtures, proteomics largely requires simplification strategies. One common approach to reduce sample complexity is to target a particular amino acid in proteins or peptides, such as cysteine (Cys), with chemical tags in order to reduce the analysis to a subset of the whole proteome. The present work describes the synthesis and the use of two new cysteinyl tags, so-called cysteine-reactive covalent capture tags (C3T), for the isolation of Cys-containing peptides. These bifunctional molecules were specifically designed to react with cysteines through iodoacetyl and acryloyl moieties and permit efficient selection of the tagged peptides. To do so, a thioproline was chosen as the isolating group to form, after a deprotection/activation step, a thiazolidine with an aldehyde resin by the covalent capture (CC) method. The applicability of the enrichment strategy was demonstrated on small synthetic peptides as well as on peptides derived from digested proteins. Mass spectrometric (MS) analysis and tandem mass spectrometric (MS/MS) sequencing confirmed the efficient and straightforward selection of the cysteine-containing peptides. The combination of C3T and CC methods provides an effective alternative to reduce sample complexity and access low abundance proteins. Copyright © 2009 John Wiley & Sons, Ltd. [source] Affinity capture using chimeric membrane proteins bound to magnetic beads for rapid ligand screening by matrix-assisted laser desorption/ionization time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2009Christian Legros The rapid and specific detection of therapeutically important ligands in complex mixtures, that may bind to membrane proteins, remains challenging for many research laboratories and pharmaceutical industries. Through its use in the development of screening assays, mass spectrometry (MS) is currently experiencing a period of tremendous expansion. In the study presented here, we took advantage of the remarkable stability properties of a bacterial membrane protein, the KcsA K+ channel, produced in E. coli and purified as a tetrameric protein in the presence of a detergent. This membrane protein can subserve as a molecular template to display the pore-forming region of human K+ channels, which are considered as targets in the search for inhibitory ligands. The engineered chimeric proteins were linked to metal-bound magnetic beads, for the screening of complex peptide mixtures, such as that of scorpion venoms. The affinity-captured scorpion toxins were eluted prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and to nano-electrospray ionization tandem mass QqTOF mass spectrometry (MS/MS) analysis. The de novo sequence of the toxins was deduced by combining the MS/MS fragmentation of the reduced form (up to the 33 first residues) and the trypsin digest peptides of the native toxins. This affinity-capture screening assay led to the isolation and characterization of potent and specific ligands of the human K+ channel, Kv1.3. The affinity-capture procedure is fast and reproducible. When linked to magnetic beads, the chimeric membrane protein can be re-used several times without losing any of its selectivity or specificity. This assay also benefits from the fact that it requires minimal amounts of animal venoms or complex mixtures, which can be expensive or difficult to procure. Copyright © 2009 John Wiley & Sons, Ltd. [source] On-line preconcentration and quantitative analysis of peptide hormone of brain and intestine using on-column transient isotachophoresis coupled with capillary electrophoresis/electrospray ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 23 2008Shifei Xia A new approach was described to achieve very sensitive analysis of peptide hormone of brain and intestine by capillary electrophoresis coupled with a transient isotachophoresis (tITP) preconcentration method. The system used was electrospray ionization mass spectrometry (ESI-MS) as detector and equipped with a sheath flow configuration. The effects of sample matrix, pH and concentration of leading electrolyte (LE), sample injection time, and ESI-MS instrumental parameters on the efficiency of the sample stacking were investigated in detail. Under the optimized conditions, lower than micromole (0.01,µM) concentrations of the peptides were easily detected. Compared to traditional hydrodynamic injection methods, about 40,230-fold increase in detection sensitivity was obtained by this technique. A distinguishing feature of the described approach is that the background electrolyte can serve as terminating electrolyte (TE), which simplifies the process of the experiments. The method was further evaluated by the analysis of low concentration active peptide mixtures spiked in hypothalamus tissue of the rat. Copyright © 2008 John Wiley & Sons, Ltd. [source] Ion trap mass spectrometry in the structural analysis of haemoglobin peptides modified by epichlorohydrin and diepoxybutaneRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 9 2002Nadia Miraglia Ion trap mass spectrometry has been shown to be particularly suitable for the structural analysis of high molecular weight peptides directly fragmented in the mass analyser without needing further sub-digestion reactions. Here we report the advantages of using multi-stage ion trap mass spectrometry in the structural characterisation of haemoglobin alkylated with epichlorohydrin and diepoxybutane. Alkylated globins were digested with trypsin and the peptide mixtures were analysed by MS3. This technique allows the sequential fragmentation of peptides under analysis, giving rise to MS3 product ion spectra with additional information with respect to MS2 mass spectra. The results obtained complete the previously reported structural characterisation of alkylated haemoglobin, demonstrating the potential of ion trap mass spectrometry. Copyright © 2002 John Wiley & Sons, Ltd. [source] Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix-assisted laser desorption/ionization-based proteomic analysisBIOMEDICAL CHROMATOGRAPHY, Issue 6 2009Atsushi Intoh Abstract Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC-HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC-HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC-HILIC has better peptide fractionation ability. We further demonstrated that ZIC-HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC-HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses. Copyright © 2009 John Wiley & Sons, Ltd. [source] A Novel MALDI Matrix for Analyzing Peptides and Proteins: Paraffin Wax Immobilized Matrix,CHINESE JOURNAL OF CHEMISTRY, Issue 1 2009Yuanlong WEI Abstract A new kind of MALDI matrix, termed paraffin wax immobilized matrix, was used to study peptide mixtures and proteins. During the preparation process, the paraffin wax was heated and coated on the stainless-steel target plate, and then 2,5-dihydrobenzoic acid (DHB) was deposited on the paraffin layer and stainless-steel target plate to obtain different kinds of matrix spots. The morphology of matrices on different supports and peptide-matrix co-crystallization were observed by a high resolution digital-video microscopy system. Peptide mixtures and bovine serum albumin (BSA) digests were used to investigate the performance of the immobilized matrices on the paraffin target. The MALDI-FTMS analysis results also showed that the detection sensitivity of matrices immobilized in the paraffin sample support was better than that on other sample supports. [source] CE-MS method development for peptides analysis, especially hepcidin, an iron metabolism markerELECTROPHORESIS, Issue 15 2009Gaëlle B. Martin Abstract A method for the resolution of a peptides mixture including hepcidin-25, an iron metabolism marker, was developed by CE-ESI-MS. Several strategies were tested to optimize peptide separation, such as the addition of cyclodextrins or organic solvents in the BGE or the use of coated capillaries. Best results in terms of resolution, symmetry and efficiency were obtained with a BGE made of 500,mM ammonium acetate pH 4.5/ACN 70:30,v/v. Using the methodology of experimental design, BGE concentration, sheath liquid composition and MS-coupling parameters were then optimized in order to obtain the best signal intensity for hepcidin. Finally, a 225,mM BGE and a sheath liquid composed of isopropanol/water 80:20,v/v containing 0.5%,v/v formic acid were selected as it constitutes the best compromise for selectivity, peak shape and sensitivity. [source] | ||||||||||